Edible hyaluronic acid‐rich burger separator discs prepared from slaughterhouse waste

Abstract In this study, edible films from chicken feet extract (CF), ovine muscle fascia extract (MF), and bovine bone gelatin powder (Gel) were prepared and their characteristics were analyzed. We also used the films as separators of burger cuts and evaluated the organoleptic characteristics of cooked burgers. Hyaluronic acid quantities of CF and MF were measured using colorimetric and spectrophotometry. Results indicated that the concentration of hyaluronic acid in CF (124.11 ppm) was greater than MF (101.11 ppm). The antioxidative property of the CF film (18.47%) was greater than the Gel (1.88%) and MF (Undetectable) films. The CF film was more resistant to water vapor permeability (2.75 × 10−9 g/m.s.pa) than the MF (1.57 × 10−8 g/m.s.pa) and Gel (1.5 × 10−7 g/m.s.pa) films. The Gel film had more appropriate mechanical properties than CF and MF films. The films kept burgers patties independent from one another and prevented them from sticking and freezing together. MF and CF films were able to promote the organoleptic properties of cooked burgers in taste and texture.

important to focus on the use of by-products in the poultry industry. Approximately 3.9 million metric tons/year of chicken feet are produced by the poultry processing industries (Chakka et al., 2017).
Chicken feet collagen primarily comprises type I and type II collagen and also contains 8.70% praline, 16.30% glycine, and 14.15% hydroxyproline (Dhakal et al., 2018). According to Lee et al. (2015), chicken feet protein film had a tensile strength (TS) of 7.13 MPa and elongation of 21.78% (Lee et al., 2015). As a result, chicken feet extract seems to be an appropriate material for making edible films.
In mammals, Collagen encompasses 25%-30% of the total body protein content. Collagen is also an important constituent of muscle tissue (forming 1%-2% of muscle tissue) where it is a chief component of the endomysium (Silvipriya et al., 2015). Moreover, fascia, which is a continuous viscoelastic tissue synthesized from layers of dense connective tissue (collagen types I and III) and interfaced by loose connective tissue, has a prominent viscoelastic property (Cowman et al., 2015). Deep fasciae can be divided into two major categories: the epimysial fasciae and the aponeurotic fascia (Fede et al., 2018). The epimysial fasciae consist of all the connective tissues which surround and interpenetrate the muscles and tendon, and are tightly adherent to them, such as epimysium, perimysium, and endomysium. The aponeurotic fascia is fibrous connective tissue layers that cover muscles and connect different segments at a distance (Fede et al., 2018). This tissue is one of the by-products of discarded sheep muscles. To date, waste from various animals has been used to produce edible films, but in this study, for the first time, aponeurotic fascia extract was used as a rich source of collagen and hyaluronic acid to prepare an edible film. Chicken and ovine by-products, such as chicken feet and ovine muscle fasciae, have considerable amounts of collagen and hyaluronic acids (Hashim et al., 2014).
The incomplete hydrolysis of collagen during moist heating produces gelatin, the use of gelatin films for food preservation applications such as in coatings or films, has been broadly researched (Luo et al., 2022). Despite the very hydrophilic nature of gelatin, gelatin films have been demonstrated to have very good process ability and they possess appropriate barrier and mechanical properties.
Moreover, the origin of the gelatin and its film-processing features have a substantial impact on the operational features of the ensuing gelatin-based films (Said et al., 2021).
One of the applications of edible films is to use them as separators between food pieces such as burgers. In the commercialization of burger products, the breaking of slices during separation just before consumption is a major problem. Slice separator films are commonly used to avoid sticking, such as oriented polypropylene, PET, or paper coated with PVDC dispersions or PE (Schneider et al., 2010). Slice separator edible films could be a good alternative to commercial separators due to the lack of negative interactions with the product and the probable good acceptance of consumers.
However, the information about the application of edible films or coatings as separator layers is very limited (Cruz-Diaz et al., 2019).
The aim of this study was to prepare films using chicken foot extract (CF) and sheep muscle fascia (MF) and to determine the amount of hyaluronic acid in them. In this study, the physical, mechanical and chemical properties of the films prepared were compared with bovine gelatin powder film (as a typical film). In addition, in order to apply the films made, they were used as burger separators and the organoleptic properties of burgers were evaluated.

| Sample preparation
Frozen chicken feet (rooster) and ovine muscle fascia (male) were purchased from the slaughterhouse. The samples were immediately transferred to the lab and were washed thoroughly to remove any contaminations, and later chopped into 3-5 cm 2 pieces. The pieces were then stored in a freezer (−20°C) separately. Gelatin powder made from bovine bone was also purchased.

| Extraction
For extract preparation, the CF and MF samples were defrosted at 4-5°C. Approximately 1000 g of CF and MF was combined with 5000 ml of water separately, and then boiled for 5 h. Afterwards, the extractions were filtered and autoclaved at 121°C for 20 min, then cooled at room temperature (23°C).

| Standards and sample preparation
According to the Dong et al. (2014) method, to obtain the reference solution 50 mg of analytical grade d-glucuronic acid (Merck KGaA) was dissolved in 100 ml of distilled water (500 μg/ml). Varying volumes of reference solution 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml were poured into the test tubes separately, and then distilled water has added until the final volume of each tube is 1.0 ml. The test tubes were then cooled to 4°C by placing them into ice water. 5 ml of freshly prepared sodium sulfate with sodium borate (Merck KGaA) was added to each test tube (4.77 g disodium tetraborate of analytical grade in 500.0 ml sulfuric acid of high-grade purity). The test tubes were sealed with caps and subsequently shaken then heated in a boiling water bath for 10 min.
Afterwards, they were cooled to room temperature and 1.25 ml of carbazole (Merck KGaA) solution (0.125 g of carbazole in 100.0 ml of absolute alcohol) was added to each test tube. The test tubes were resealed, shaken and heated again for 15 min in a hot water bath (100°C).
Later, they were cooled down in an ambient condition, and the color of the solution changed to purple. We measured the absorbance of so-

| Film formation
The films were prepared using a casting method; 100 ml of the CF and MF's liquid extract was mixed with the concentration of glycerol as plasticizer (5% w/v). For the preparation of the gelatin film, 10 g of dry matter and 1 g of glycerol were dissolved into 100 ml of boiling water. Approximately 10 ml of each film solution was poured into an 8-cm Petri dish and dried at 37°C for 18 h. The dried films were placed in plastic zipper bags and stored in the refrigerator prior to testing.

| Film thickness
The thickness of the films was measured using a digital micrometer Tokyo,Japan) with exactness of 1 μm at 10 random positions on the film. The obtained thickness values were applied for calculating the films' water vapor permeability and tensile properties.

| Moisture content
The moisture content of the films was measured by drying the samples in an oven at 105°C until the weight remained constant. The weight loss of samples was determined before and after drying with a scale accuracy of 0.001 g (Khodaei et al., 2020).

| Solubility in water
Using the method outlined by Tongdeesoontorn et al. (2012), the water solubility of the films was determined. Pieces of the films (3 cm × 3 cm) were dried in an oven (105°C for 5 h). Then, the films were placed in a beaker with 30 ml of distilled water and they were shaken for 24 h at 25°C. The undissolved remnants were filtered and dried at 105°C for 5 h (Tongdeesoontorn et al., 2012).

| Water vapor permeability (WVP)
In line with the E96-00 method, we determined the WVP of films gravimetrically. The film samples with an effective area of 31.4 mm 2 were situated on test cups which each contained 3.0 g of anhydrous sodium chloride (0% relative humidity, RH, assay cup) and were then sealed. Each cup was placed in a desiccator containing a saturated solution of sodium chloride at 25°C. The weight of the cups was measured throughout 3 h intervals for 48 h (Standard ASTM, 1989). The WVP was calculated using the following formula: where (A) represents the area of exposed film surface in m 2 , (Δm/ Δt) is the weight of moisture gain per unit of time (g/s), (X) represents film thickness m, and (Δp) is the difference of water vapor pressure between two films.

| Tensile strength (TS) and elongation at break (EAB)
To evaluate the mechanical properties of the films, we utilized their tensile strength (TE) and Elongation at Break (EAB) as proxy measures. These mechanical qualities were evaluated at 25°C and 50% RH. In this study, we utilized the D882-18 standard test method and the H5KS Stable Micro System, UK, was used as the testing instrument. The films were conditioned in 50% RH in a desiccator containing saturated solutions of Mg(NO 3 ) 2 for 48 h. Film samples measuring 20 mm × 100 mm, were cut from each film and were located between the grips of the testing instrument. The initial grip distance and the cross-head speed were set at 50 mm and 5 mm/min, respectively (ASTM International, 2012).

| Scanning electron microscopy (SEM)
To determine the film's microstructure, we utilized Scanning Electron Microscopy SEM (EM-3200, KYKY). The films were frozen in liquid nitrogen and fractured. After that they were mounted onto aluminium stubs with double-sided tape, and then were coated with a thin layer of gold using a BAL-TEC SCD 005 sputter coater (BALTEC AG, Balzers, Liechtenstein). Low pressure and an accelerating voltage at 20 kV were used for SEM imaging.

| FTIR spectra of the films
The infrared spectrum of absorption or emission of a matter was determined using Fourier Transform Infrared Spectroscopy (FTIR) (Thermo Nicolet, Avatar 370). At first, the films were powdered to set up the discs, and then 70 mg of spectroscopic grade KBr was mixed fully with roughly 2 mg of the film's powder. Subsequently, the powder was hard-pressed into pellets to obtain a transparent disc 15 mm in diameter and 0.54 mm in thickness. The FTIR spectra were obtained in the 4000-400 cm −1 range, at 25°C, by co-adding 32 scans with 4 cm −1 spectral resolution.

| Differential scanning calorimeter (DSC)
The thermal properties of films were analyzed with Mettler Toledo, DSC-1, Switzerland. Samples (approx. 7 mg) were weighed into the reference (an empty aluminium pan), and heating rate was programmed by setting the heater at 10°C/min between a range of −100°C and 200°C. Glass transition temperatures (T g ) and melting temperatures (T m ) of each film were established from resulting thermo grams as the midpoint temperature of the shift in the baseline due to the change in the heat capacity upon the glass transition.
Readings of T g were taken twice and the average of the results have been presented.

| Radical scavenging activity of the films
The DPPH radical scavenging activity was carried out according to the procedure of Brand-Williams et al. (1995) (Brand-Williams et al., 1995). A quantity of 25 mg of the sample was dissolved in 5 ml of distilled water. Later, 0.1 ml of the solution was mixed with 3.9 ml of the DPPH solution (0.1 mM methanol solution). They were then incubated in the dark for 30 min at 25°C. Whilst mixing the DPPH, a stable non-radical form of DPPH was obtained with simultaneous change of the violet color to a pale yellow. The Perkin-Elmer spectrophotometer was used to measure absorbance at 517 nm. We determined the percentage of DPPH radical-scavenging activity using the following equation:

| Sensory analysis
Burgers (50 g) were prepared manually with a round-shaped mold.
Burgers were separated with films aseptically at room temperature.
Fresh burgers without films were used as controls. All samples were placed in trays covered with aluminium foil and stored at −20°C until analysis. Sensory analysis was performed by a group of 5 trained panelists using a 5-point hedonic scale ranging from "very strong like, score 5" to "very strong dislike, score 1." A score of 1-5 was assigned for the overall acceptability of the cooked samples (control, and wrapped), which was determined by assessing the appearance, color, odor, taste, texture, and flavor (Stone & Sidel, 2004).

| Statistical analysis
All tests were performed three or more times. We have presented the data as mean values with their standard deviation and these values were used in the statistical analysis. Significant differences were found by one-way ANOVA and the means were compared using Duncan's multiple range test p < .05. We used statistical software SPSS (Inc., Chicago, IL, Ver. 21) to carry out the statistical analysis.

| Proximate composition of extractions
The proximate composition of the extraction obtained from CF and MF in relation to protein, lipid, ash and moisture content, is presented in Table 1. The moisture content verified was 8.67% for CF, very close to that found by Almeida and Lannes (2013) of 9.74% for chicken feet gelatin and the moisture content of MF was 8.56%. As seen in Table 1, the CF and MF extract presented 1.66% ash. The CF protein content is 78.96%, but the MF has a higher protein content (77.28%). About the content of lipids, CF was 10.71%. Almeida and Lannes (2013) reported the presence of 12.8% lipid in chicken feet.
The lipid content of MF extract was 12.5%.

| Determination of hyaluronic acid concentration
Based on the equation obtained from the standard diagram, the amount of hyaluronic acid in CF extract was 124.11 and in the extract obtained from MF was 101.40 ppm. The data show that the amount of hyaluronic acid in CF extract is significantly higher than in the MF extract. According to previous studies, the rooster comb has the highest amount of hyaluronic acid in living tissues. The amount of this substance in the rooster comb is about 7500 μg/mL (Kanchwala et al., 2005). This amount is about 60 times more than  (Li et al., 2020). Nevertheless, the use of poultry and sheep by-products are not only inexpensive and more accessible than many sources, but also have a significant amount of hyaluronic acid, which can be used in food and packaging industries. The extracts of CF and MF generated in this work could simply result in homogeneous solutions, which provide translucent films that can be readily manipulated (Figure 1).

| Thickness
As can be seen in Table 3, the average thickness for the CF and MF films is 0.10 mm and for the Gel film is 0.17 mm which is significantly larger than the other films. Hanani et al. (2019) reported that the thickness of their fish gelatin film was 0.06 mm and Li et al. (2020) reported the thickness of their B type gelatin film to be 0.07 mm (Hanani et al., 2019;Li et al., 2020). As such, these materials were thin enough to be considered as films, and the effect of the observed differences in thickness on the film's functional properties could be negligible. Controlling the film thickness is an essential factor which may influence the mechanical, barrier, and transparency properties of the films (Mir et al., 2018). Moreover, discrepancy in film thicknesses may result from the type of solid matter used as well as its amount. The film preparatory methods and drying conditions may also influence the thickness (Galus & Lenart, 2013).

| Moisture content
According to the data presented in Table 3, a significant difference was found between the average moisture content of the CF and Gel films (7.40% and 7.45%, respectively), and the MF film (5.90%).
The moisture content of chicken feet was reported to be 65.08% in the study (Hashim et al., 2014). Lee et al. (2015) also reported that the moisture content of chicken feet protein film with glycerol and sorbitol as plasticizer to be 10.17% and Hanani et al. (2019) reported that the moisture content of a fish gelatin film was 11.05% (Hanani et al., 2019;Lee et al., 2015). The higher humidity in CF and Gel films are dependent on its hydrophilic nature which is mostly due to the tendency to form hydrogen bonds with water molecules.
The reduction in moisture content is mainly related to the hydrophobicity of the fats present in the film components with gelatin base, which greatly reduces the film water absorption capacity (Li et al., 2020).

| Solubility in water
The solubility percentages of the films' after 24 h immersion in water has been detailed in Table 3. This was mesured to be 99.09% for the MF film, 68.49% for the CF film, and 41.62% for the Gel film.
After 24 h of incubation in water, the CF and MF films changed shape while the gelatin film completely retained its structure. Specifically, the solubility of MF films (99.08%) was significantly higher (p < .05) and the solubility of the Gel film (40.65%) was significantly lower (p < .05) than the other films. In one study, the film solubility levels for chicken skin gelatin was 94% (Loo & Sarbon, 2020),  Table 3 shows the WVP of the films. According to these results, the CF film had significantly lower WVP (2.75 × 10 −9 g/m.s.Pa) than the MF film (1.57 × 10 −8 g/m.s.Pa) and the Gel film (1.5 × 10 −7 g/m.s.Pa).

| Water vapor permeability (WVP)
this data is consistent with findings by Lee et al. (2015) where they reported the WVP of chicken feet protein film without plasticizer to be 3.44 × 10 −9 g m/m 2 s Pa (Lee et al., 2015). In another study, Li et al. (2020), reports that the WVP of gelatin (type B) film to be

| Contact angle measurements
The contact angle is one of the most frequently used measures when assessing surface properties of biopolymers. It provides information regarding films' surface wetting or non-wetting prop- tend to have contact angles below 90° (Bracco & Holst, 2013).

erties. The results have been demonstrated in
Accordingly, the two films of CF and MF were hydrophobic, but the Gel film was hydrophilic. As shown in

| Tensile strength (TS) and elongation at break (EAB)
The TS and EAB of the films have been demonstrated in Table 2.
The maximum tensile stress a film can sustain corresponds to its TS, whereas the maximum change in length of a test specimen before breaking is defined as EAB (Pereda et al., 2012). Mechanical strength is a general necessity to retain the integrity of packaging films and to be able to withstand external stress (Yang & Paulson, 2000). The TS of the Gel film (5.7 MPa) was significantly greater (p < .05) than the MF (3.9 MPa) and CF (2.49 MPa) films. Lee et al. (2015) reported that the TS of chicken protein film with sorbitol was 3.38 MPa and Hanani et al. (2019) reported that the TS of a pure fish gelatin film was 7.22 MPa (Hanani et al., 2019;Lee et al., 2015). The strength and weakness of the hydrogen bonds of the film molecules determine the TS, so the stronger the internal network and the cohesion of the film, the higher the TS. The increase of TS may also be due to Note: All measurements were performed at 25°C and RH = 50%. Different letters in the same column indicate significant difference (p < .05).
differences in molecular weight, size, number of oxygen atoms, and hydrophilicity of the film matrix molecules.
Among the different films, the CF film had a greater EAB (89.05%) compared to the MF (85.25%) and Gel (70.50%) films, respectively.
In contrast to the TS, the EAB of CF film increased significantly, implying that the film was more pliable when compared to the gelatin film. The occurrence of lipid in CF films resulted in increased EAB of films being observed (Table 3). It appears as though the presence of lipid globules throughout the film matrix leads to a decrease in

| Scanning electron microscopy
The films' surface morphology was investigated by scanning electron microscopy ( Figure 2). The cross-linked Gel film was observed to be uniform, compact and homogenous in appearance, while the microstructures of the MF film contained some bubbles. The surface of the CF film was rougher than the MF and Gel films. The increase in surface roughness of CF film is attributed to the migration of fat droplets towards the film surface during the film drying process.

| FTIR spectra of the films
FTIR spectroscopy has previously been extensively used for the identification of intermolecular interactions in polymers (Liu
Abbreviations: CF, Chicken feet film; Gel, Bovine bone gelatin film; MC, moisture content; MF, muscle fascia film; WS, water solubility; WVP, water vapor permeability. with previous studies (Hanani et al., 2019). Generally, similar spectra for the three types of films were observed.

| Differential scanning calorimeter (DSC)
Several phase transitions can occur when polymer materials are thermally processed, and each transition relates to a certain thermal property. The glass transition temperature (T g ), which is defined as "as the temperature at which a polymer undergoes a structural transition from a glassy to a rubbery state" is one of these transitions.
Under the T g value, the films are rigid, stiff, and glassy, in comparison to levels greater than T g , where the films become elastic and soft (. Yang & Paulson, 2000). Additionally, there is the melting transition (T m ), where at a temperature known as the melting temperature, there is a transition from a crystalline to an amorphous phase, a liquid-like state (Alavi et al., 2014). The results showed only one T g for the films (Table 2). Corresponding to Ghasemlou et al. (2011), if a plasticizer is not homogenized with a polymer, the mixture would have two T g values, corresponding to two pure phases (Ghasemlou et al., 2011). The glass transition, melting and deterioration peaks of the films prepared from CF, MF, and Gel films were around 33. 80-35.42°C, 28.54-30.36°C, and 38.55-40.53°C, respectively.
From the gelatin film, the T g results reported may be explained by the block copolymer model for the amino acid content of gelatin. The T g of gelatin takes place at ~60°C and is related with the T g of its amino acid in the peptide chain. Additionally, native fish gelatin film was found to have a T m of 76.5°C, which is found to be higher than films 91°C (Bigi et al., 2001). The variation in observed thermal properties is appreciable due to different versions of the gelatin source used.
Moreover, Bigi et al. (2004) described that the initial transition is likely correlated with the evaporation of absorbed water in the gelatin sample, and a higher transition temperature is associated with the relative quantity of the triple helix in gelatin. As such, thermal properties of films are influenced by their source (Bigi et al., 2004).

| Sensory analysis
Results of the organoleptic evaluation of hamburger samples (control, and separated with films) have been presented in Figure 4.
Compared to the control samples, the burgers with the film were easily separated from each other after freezing. During defrost, the films kept their shape and covered the burgers well ( Figure 5).
The MF film retained its clarity and uniformity while the CF and gel films were wrinkled during freezing. When cooking, the films were completely covered on the burgers. After cooking, the taste, odor, texture, and overall acceptance of burgers with MF and CF films revealed a higher score than the sample with the gel film and control.
The presence of films on the surface of the burger seems to prevent the loss of moisture during cooking and creates a more juicy texture in the burger. The heat also melted the film and penetrated into the texture of the burger, which, due to the nature of the CF and MF films, helped to create a more meaty taste.

| CON CLUS ION
Chicken feet and ovine muscle fascia extracts can be used as a precursor to edible films. Although the concentration of hyaluronic acid in CF was more than in the MF extract, the amount of hyaluronic acid in both extracts was acceptable. The CF film had better WVP, and antioxidant properties than the MF and Gel films.
The physical, thermal, and mechanical properties varied depending on the type of film source. It appears that the TS of the Gel film was more acceptable than the CF and MF films. FTIR results clearly indicated intermolecular interactions between film components. These interactions are related to the presence of amine, hydroxyl, and/or carboxylic groups in the polysaccharides (hyaluronic acid) and collagen in the CF and MF films. Accordingly, the different mechanical and thermal properties of biopolymer films can be a result of hydrogen bonds between the reactive groups of components. From these results, it can be concluded that CF film could be an appropriate packaging material applied to food products. In relation preliminary study of CF and MF films for the utilization as separator for burger slices, promising results were obtained and further research is warranted.

ACK N OWLED G M ENT
We gratefully acknowledge the support of the Neyshabur University of medical science for funding this project (project number 97284, code of ethics IR.NUMS.REC.1398.002).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.